Immunologic effector cell of targeted CLD18A2, and preparation method and use thereof

ABSTRACT

Disclosed are a chimeric antigen receptor (CAR) targeting CLD18A2, and preparation method and use thereof. The extracellular binding region of the CAR comprises a protein specifically recognizing CLD18A2. The immune effector cell modified by the CAR can be used to treat tumors such as pancreatic cancer and stomach cancer.

CROSS-REFERENCE

This application is a continuation of U.S. patent application Ser. No.15/326,974, filed Jan. 17, 2017, now U.S. Pat. No. 10,377,822, which isa national stage entry of International Patent Application No.PCT/CN2015/084023, filed Jul. 15, 2015, which claims priority to ChinesePatent Application No. 201410341504.X, filed Jul. 17, 2014, each ofwhich is entirely incorporated herein by reference.

TECHNICAL FIELD

The invention belongs to the field of cell therapy for tumor, andparticularly relates to an immune effector cell targeting CLD18A2, apreparation method and application thereof.

TECHNICAL BACKGROUND

Attention has been increasingly paid to the role of T lymphocytes intumor immune responses. The Adoptive immunotherapy based on Tlymphocytes has a certain effect in some tumors, moreover, such immunetherapy method can overcome the above defects of antibody treatment,however, the therapeutical effect in most tumors is still notsatisfactory [Grupp S A, et al. Adoptive cellular therapy. Curr TopMicrobiol Immunol., 2011; 344:149-72]. In recent years, based on thediscovery that the identification of a target Cell by CTL isspecifically dependent on a T lymphocyte receptor (T Cell receptor,TCR), the scFv of the antibody against tumor cell-related antigen isfused to intracellular signal activation motif such as T-lymphocytereceptor CD3ζ or FcεRIγ to form Chimeric antigen receptors (CAR), andcan be genetically modified on the surface of T lymphocyte by means suchas lentivirus infection. Such CAR T lymphocyte can selectively direct Tlymphocyte to tumor cells and specifically kill tumor cells in a majorhistocompatibility complex (MHC)-independent manner. CAR T lymphocytesare new immune therapy strategy in the tumor immunotherapy field[Schmitz M, et al. Chimeric antigen receptor-engineered T cells forimmunotherapy of Cancer. J Biomed Biotechnol, 2010,doi:10.1155/2010/956304].

Chimeric antigen receptor comprises an extracellular binding domain, atransmembrane region and an intracellular signaling domain. Generally,the extracellular domain comprises an scFv that is capable ofrecognizing a tumor-associated antigen, the transmembrane region employsthe transmembrane region from molecules such as CD8, CD28 and the likes,and the intracellular signaling domain employs an immunoreceptortyrosine-based activation motif (ITAM) CD3ζ or FcεRIγ and theintracellular signaling domain of co-stimulatory signaling molecule suchas CD28, CD27, CD137, CD134 and the likes.

In the first generation CAR T lymphocyte, the intracellular signalingdomain comprises ITAM only, and parts of the chimeric antigen receptorare connected in the form of scFv-TM-ITAM. Such CAR T can inducecellular cytotoxic effect against tumor, but the level of cytokinessecreted is relatively low, and no sustaining anti-tumor effect could beinduced in the body (Zhang T. et al., Chimeric NKG2D-modified T cellsinhibit systemic T-cell lymphoma growth in a manner involving multiplecytokines and cytotoxic pathways, Can Res 2007, 67 (22): 11029-11036).

In the second generation CAR T lymphocyte that developed afterwards, anintracellular signaling domain of CD28 or CD 137 (also known as 4-1BB)is further included, and parts of the chimeric antigen receptor areconnected in the form of scFv-TM-CD28-ITAM or scFv-TM-/CD137-ITAM.Co-stimulatory effect of B7/CD28 or 4-1BBL/CD137 in the intracellularsignaling domain induces sustained proliferation of T lymphocytes, andis capable of increasing the level of cytokines such as IL-2, IFN-γ andothers secreted by T lymphocytes, as well as improving the in vivosurvival period and the anti-tumor effect of the CAR T (Dotti G. et al.,CD28 costimulation improves expansion and persistence of chimericantigen receptor modified T cells in lymphoma patients. J Clin Invest,2011, 121 (5): 1822-1826).

In the third generation CAR T lymphocyte that developed in recent years,parts of the chimeric antigen receptor are connected in the form ofscFv-TM-CD28-CD137-ITAM or scFv-TM-CD28-CD134-ITAM, the in vivo survivaland the anti-tumor effect of the CAR T is further improved (Carpenito C,et al., Control of large established tumor xenografts with geneticallyretargeted human T cells containing CD28 and CD 137 domains, PNAS, 2009,106(9): 3360-3365).

Besides the attractive prospect of CAR T lymphocyte in tumorimmunotherapy, its relatively high risk shall be taken into account. Forinstance, certain normal tissue(s) may exhibit low expression ofspecific antigen to be recognized by the CAR, this may results in damageby CAR T lymphocytes to such normal tissues. For example, treatmentagainst carbonic anhydrase IX (CAIX), the antigen expressed in tumorcells of patients having renal cell carcinoma, is the first reportedcase of clinical application of adoptive therapy with CAR T lymphocytes,which is also the first case reporting on-target off-tumor effect of CART lymphocytes. After multiple administrations of CAR T lymphocytes,patients developed liver toxicity of grades 2-4. Upon analysis, thecause is believed to be the CAIX expression in a low level on bile ductepithelial cells, this clinical trial was discontinued while assessmentabout therapeutic outcomes in patients are excluded (Stoter G. et al.,Treatment of metastatic renal cell carcinoma with autologousT-lymphocytes genetically retargeted against carbonic anhydrase IX:first clinical experience, J clin oncol, 2006, 24 (13): e20-e22; Ngo MC, et al., Ex vivo gene transfer for improved adoptive immunotherapy ofcancer Human Molecular Genetics, 2011, R1_R7). Furthermore, theexcessive co-stimulation signal in CAR may reduce the threshold requiredfor activating effector cells, such that genetically modified Tlymphocyte may be activated at conditions of rather low level of antigenor at the absence of antigen pulse, and resulting in the release oflarge amount of cytokines which may induce so-called “cytokine storm”.This signal leakage will cause off-target cytotoxicity, resulting innon-specific tissue damage. For example, sudden death of a patientcaused by such “cytokine storm” induced by low Her2 expression in normallung tissue was observed during a clinical treatment using athird-generation CAR T cells targeting Her2 for patients having advancedcolorectal cancer with liver and lung metastasis (Morgan R A, et al.,Report of a serious adverse event following the administration of Tcells transduced with a chimeric antigen receptor recognizing Erbb2Molecular Therapy, 2010, 18 (4): 843-851).

When CAR T is designed, selection of the antigen gene target is crucial.Because of the complexity of in-vivo gene expression and variousuncontrollable factors, it is extremely difficult to select a suitablegene for CAR T. Furthermore, for many tumor-specific antigens, it isvery difficult to find specific molecule directing at it and suitable toconstruct CAR-modified immune effector cell. After the CAR T isestablished, it is often unable to obtain an active extracellularbinding region, which is also a difficulty for developing CAR Ttechnology.

SUMMARY OF THE INVENTION

The object of the present invention is to provide an immune effectorcell targeting CLD18A2 and the preparation method and use thereof.

In a first aspect of the present invention, it is provided chimericantigen receptor (CAR) expressed on the surface of an immune effectorcell, wherein the chimeric antigen receptor comprises sequentiallyconnected extracellular binding region, transmembrane region andintracellular signal region, wherein the extracellular binding regioncomprises protein which specifically recognizes CLD18A2 (claudin18.2).

In one preferred embodiment, the protein specifically recognizingCLD18A2 is an antibody or a ligand; preferably, the antibody is asingle-chain antibody or a domain antibody.

In another preferred embodiment, the transmembrane region is a sequencecomprising transmembrane regions and hinge regions of CD8 or CD28.

In another preferred embodiment, the intracellular signal region isselected from the intracellular signal region sequence of the following:CD3ζ, FcεRIγ, CD27, CD28, CD137, and CD134, and a combination thereof.

In another preferred embodiment, the chimeric antigen receptor comprisesan extracellular binding region, a transmembrane region and anintracellular signal region connected in the following sequence:

Single chain antibody specifically recognizing the CLD18A2, CD8 and CD3;

Single chain antibody specifically recognizing CLD18A2, CD8, CD137 andCD3;

Single chain antibody specifically recognizing CLD18A2, transmembraneregion of CD28(CD28a), intracellular signal region of CD28 molecule(CD28b) and CD3; or

Single chain antibody specifically recognizing CLD18A2, transmembraneregion of CD28, intracellular signal region of CD28, CD137 and CD3.

In another preferred embodiment, the chimeric antigen receptor comprisesany one of the amino acid sequence of SEQ ID NO: 19-22.

In another preferred embodiment, the immune effector cell comprises Tlymphocytes, NK cells or NKT cells.

In another aspect of the invention, it is provided the nucleic acidencoding the chimeric antigen receptor.

In one preferred embodiment, the nucleic acid comprises any one of thenucleotide sequence of SEQ ID NO: 15-18.

In another aspect of the present invention, it is provided an expressionvector comprising the aforementioned nucleic acid.

In one preferred embodiment, the expression vector is derived fromlentivirus plasmid PWPT (or PWPT-eGFP).

In another aspect of the present invention, it is provided a virus,wherein the said virus (such as lentiviral vector) comprising saidvector.

In another aspect of the invention, it is provided the use of thechimeric antigen receptor, the nucleic acid, the expression vector, orthe virus, for preparing a genetically modified immune effector celltargeting CLD18A2.

In another aspect of the invention, it is provided a geneticallymodified immune effector cell transducted by said nucleic acid, saidexpression vector, or said virus.

In another aspect of the invention, it is provided a geneticallymodified immune effector cell wherein a chimeric antigen receptor isexpressed on the surface thereof, wherein the amino acid sequence of thechimeric antigen receptor is selected from any one of the amino acidsequences of SEQ ID NOs: 19-22.

In another aspect of the present invention, it is provided the use ofthe genetically modified immune effector cells for preparation of amedicine for suppressing tumor, wherein the tumor is CLD18A2 positive(high-expression) tumor.

In another preferred embodiment, the CLD18A2 positive tumor includespancreatic cancer, gastric cancer.

Other aspects of the invention will be apparent to those skilled in theart from the disclosure herein.

DESCRIPTION OF THE DRAWINGS

The patent application file contains at least one drawing executed incolor. Copies of this patent or patent application publication withcolor drawing(s) will be provided by the Office upon request and paymentof the necessary fee.

FIG. 1 is the structure schematic diagram of the present lentivirusvector pWPT-eGFP-F2A-CAR comprising CAR coding sequence.

FIG. 2 is a schematic diagram of the connection sequence of each part ofthe chimeric antigen receptor.

FIG. 3 is the electrophoresis image of the purified anti-CLD18A2 singlechain antibody of Example 1.

FIG. 4 is the results of the western blot assay of the cell lines stablyexpressing CLD18A1 and CLD18A2.

FIG. 5 is flow cytometric detection of the binding specificity ofCLD18A2 single-chain antibody with CLD18A1 and CLD18A2.

FIG. 6 is the electrophoresis identification of a spliced chimericantigen receptor.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

Through thorough and in-depth study, the inventors discloses for thefirst time a CAR modified immune effector cell based on CLD18A2 gene andthe preparation method thereof.

CLD18A2 Gene

At early stage, the inventors investigated various kinds oftumor-specific genes and found that a relatively large part of thesegenes are also expressed in part of normal tissue cells, thus unable tobe applied in the chimeric antigen receptor T cell. Some tumor-specificgenes have better tumor-specific expression characteristics, but theCAR-modified immune effector cells correspondingly designed do not haveor have rather low tumor cytotoxicity. This may be caused by that theproteins expressed by corresponding genes have low antigenicity, orexpressed on an inapposite location, or expressed at a level not highenough, etc. It is also possibly caused by the recombinant constructionprocess which weakened the tumor killing ability of T lymphocytes orcause the tumor killing ability lost.

After repeated investigation and screening, the inventors discoveredCLD18A2 gene as the target gene for designing CAR modified immuneeffector cell (for example, T lymphocyte). Claudin 18 (CLD18) molecule(Genbank accession number: splice variant 1 (CLD18A1): NP.sub.-057453,NM.sub.-016369, and splice variant 2 (CLD18A2): NM.sub.-001002026,NP.sub.-001002026) is a transmembrane protein with a molecular weight ofabout 27.9/27.72 kD. Claudins is a tightly-connected membrane proteinwhich locates on the epithelium and the endothelium.

Study shows that the CLD18A1 selectively expresses on the normal lungand stomach epithelium, while the CLD18A2 only expresses ondifferentiated cells with short lifespan in stomach epithelium, not ongastric stem cells. Meanwhile, researches have indicated that theCLD18A2 is expressed on various tumor cells. In view of theabove-described characteristics of CLD18A2, the inventors havespeculated CLD18A2 be an important therapeutic target for these tumors.Said speculation has been verified by abundant subsequent work.

Chimeric Antigen Receptor and the Coding Nucleic Acid Thereof

The present invention provides a chimeric antigen receptor expressed onthe surface of T lymphocyte, wherein the chimeric antigen receptorcomprises sequentially connected extracellular binding region,transmembrane region and intracellular signal region, wherein theextracellular binding region comprises protein that specificallyrecognizes CLD18A2 (claudin 18.2). The chimeric antigen receptor isexpressed on the surface of T lymphocyte, which make the T lymphocytehas highly specific cytotoxic effect on tumor cells which expressedCLD18A2 at a high level.

As a preferred mode of the present invention, the extracellular bindingregion comprises a single-chain antibody scFv that specificallyrecognizes CLD18A2 The extracellular binding region of theabovementioned chimeric antigen receptor protein is connected with atransmembrane region of CD8 or CD28 through a CD8 hinge region, and thecross-membrane region is immediately followed by the intracellularsignal region.

The present invention also includes nucleic acid encoding the chimericantigen receptors. The nucleic acid sequence of the present inventioncan be a DNA form or a RNA form. The DNA form comprises cDNA, genomicDNA, or artificially synthesized DNA. DNA can be single-stranded ordouble-stranded, a coding chain or a non-coding chain. The codons of thenucleic acid of the present invention coding the amino acid sequence ofthe present chimeric antigen receptor protein can be degenerate, thatis, a variety of degenerate nucleic acid sequences encoding the sameamino acid sequence are included in the scope of the present invention.The degenerate nucleic acid codons encoding corresponding amino acid arewell known in the art. The present invention also relates to variants ofthe polynucleotide, which encode polypeptides or fragments, analogs andderivatives of the polypeptides having the same amino acid sequences asthe present invention. The variants of the polynucleotide can benaturally occurring allelic variants or non-naturally occurringvariants. These nucleotide variants include substitution variants,deletion variants, and insertion variants. As is known in the art, anallelic variant is an alternative form of a polynucleotide, which may besubstitution, deletion or insertion of one or more nucleotides, but doesnot substantially alter the functionality of the polypeptide codedthereby.

The monoclonal antibody specifically recognizing human CLD18A2 can bechosen from the the antibodies disclosed in the prior art. A variety ofmonoclonal antibodies that recognize the c-terminal epitope of CLD18A2may be applied in the present invention in a suitable manner, as long asafter the recombinant construction, CAR-modified immune effector cellwith killing activity can finally be obtained. Preferably, asingle-chain antibody, more preferably the single-chain antibodies are163 and 175 antibodies; The 163 and 175 antibodies can specificallyrecognize CLD18A2 but not CLD18A1. More preferably, they are connectedto Fc (ScFv-163 and ScFv-175).

The term “single-chain antibody (scFv) fragment” as used herein refersto an antibody fragment defined as follows. It is a recombinant proteincomprising heavy chain variable region (VH) and a light chain variableregion (VL) connected by a linker, and the linker associates the twodomains by which an antigen binding site is finally formed. The size ofthe scFv is generally ⅙ of one complete antibody. Preferably,single-chain antibody is one amino acid chain sequence coded by onenucleotide chain. Single-chain antibodies used in the present inventionmay be used alone or in combination with conventional techniques knownin the art, for example, amino acid deletion, insertion, substitution,addition, and/or recombination and/or other modification methods forfurther modification. It is well known to those skilled in the art tointroduce modification in the DNA sequence according to the amino acidsequence of the antibody, for example, see Sambrook, Molecular Cloning:A laboratory Manual, Cold Spring Harbor Laboratory (1989) N.Y. Themodification is preferably performed on the nucleic acid level. Theabove single-chain antibodies can also comprise derivatives thereof. The“derivatives of antibodies” in the present invention include, forexample, derivatives of the antibodies which obtained by phage displaytechniques, and the binding efficiency of said antibodies with CLD18A2antigen epitope is increased by surface plasmon resonance technique thatused in the Biacore system (Schier, Human Antibodies Hybridomas 7(1996), 97-105; Malmborg «Journal of Immunological Methods», 1995,183:7-13). Also included are those antibody derivatives produced by thepreparation method of chimeric antibody described in for exampleWO89/09622, the humanized antibody preparation method described inEP-A10239400 and WO90/07861, the method for producing xenogeneicantibodies, for example human antibodies in mice, which is mentioned inWO 91/10741, WO 94/02602, and WO 96/33735.

The term “specific recognition” of the present invention means that theantibody of the present invention does not react with or substantiallydoes not react with any polypeptide other than a target antigen. Thedegree of specificity can be determined by immunology techniquesincluding, but not limited to, immunoblotting, immunoaffinitychromatography, flow cytometry and the like. In the present invention,the specific recognition is preferably determined by flow cytometry.Under particular conditions, the standard of specific recognition can bedetermined by those of ordinary skill in the art based on theirknowledge of the art.

The transmembrane region of the chimeric antigen receptor can beselected from the transmembrane region of proteins such as CD8 or CD28.CD8 or CD28 are the natural markers on the surface of T lymphocyte.Human CD8 protein is a heterodimer, consisted of two chains, αβ or γδ.In one embodiment of the invention, the transmembrane region is selectedfrom the transmembrane region of CD8 alpha or CD28. Additionally, theCD8 alpha hinge region (hinge)) is a flexible region. Therefore, thetransmembrane region of CD8 or CD28 and the hinge region can be used toconnect the target recognition domain scFv with an intracellular signalregion in the chimeric antigen receptor (CAR).

The intracellular signal region may be selected from the intracellularsignal regions of CD3ζ, FcεRIγ, CD28, CD137 and CD134 proteins, andcombinations thereof. The CD3 molecule consists of five subunits,wherein CD3ζ subunit (also called as CD3 zeta, for short “Z”)) comprisesa 3-ITAM motif, which is an important signal transduction region inTCR-CD3 complex. CD3δZ is a truncated CD3ζ sequence without ITAM motif,which is commonly used for constructing negative control in the practiceof the present invention. FcεRIγ is mainly distributed on both mastcells and basophilic granulocyte surfaces, which contains an ITAM motif,and is similar to CD3ζ in structure, distribution and function.Furthermore, as previously described, CD28, CD137 and CD134 areco-stimulating signal molecules, which can cause sustained proliferationof T lymphocytes by the co-stimulation action generated by theintracellular signal segments after binding with the respective ligands,and can increase the level of cytokines secreted by T lymphocyte such asIL-2 and the IFN-gamma and the like, while increasing the survival cyclein vivo and the anti-tumor effect of the CAR-modified immune effectorcells.

The anti-CLD18A2 chimeric antigen receptor protein coded by the nucleicacid of the present invention can be sequentially connected according tothe following manner:

scFv(CLD18A2)-CD8-CD3ζ,

scFv(CLD18A2)-CD8-CD137-CD3ζ,

scFv(CLD18A2)-CD28a-CD28b-CD3ζ,

scFv(CLD18A2)-CD28a-CD28b-CD137-CD3ζ,

and combinations thereof, wherein CD28a in the related chimeric antigenreceptor protein represents a CD28 transmembrane region, CD28brepresents intracellular signal region of CD28 molecules. The variousanti-CLD18A2 chimeric antigen receptors above are collectively referredto as scFv (CLD18A2)-CAR.

In one embodiment of the invention, the nucleic acid disclosed by theinvention has the sequence as shown in SEQ ID NO: 15-18. In anotherembodiment of the present invention, the nucleic acid of the presentinvention is a nucleic acid encoding the chimeric antigen receptorprotein as shown in one of SEQ ID NOs: 19-22.

Expression Vector and Cell

The present invention also provides vector comprising the nucleic acidencoding the abovementioned chimeric antigen receptor protein expressedon the surface of T lymphocyte. In one embodiment, the vector used inthe present invention is a lentivirus plasmid vector pWPT-eGFP. Saidplasmid belongs to a third-generation self-inactivated lentivirus vectorsystem, which have 3 plasmids, that is, the packing plasmid psPAX2encoding protein Gag/Pol and Rev protein; envelope vector PMD2.Gencoding VSV-G protein; and empty vector PWPT-eGFP, which can be usedfor recombinant introduction of the target nucleic acid sequence, thatis, a nucleic acid sequence encoding a CAR. In the empty vectorpWPT-eGFP (which itself is mock in the following experiments), theelongation factor-1 alpha (EF-1α) promoter regulates the expression ofthe enhanced green fluorescent protein (eGFP), while in the recombinantexpression vector encoding target gene CAR, the co-expression of eGFPand CAR is realized via ribosome skipping sequence 2A (shortened as“F2A”) from food-and-mouth disease virus (FMDV).

The present invention further comprises the virus comprising suchplasmid. The virus disclosed by the invention comprises infectious virusafter packing, as well as the to-be-packaged virus comprising essentialelement for packaging into an infectious virus. Other viruses known inthe art for transferring foreign genes into T lymphocytes and theircorresponding plasmid vectors can also be used in the present invention.

In one embodiment of the invention, the said virus is a lentiviruscomprising the above-mentioned pWPT-eGFP-F2A-CAR recombinant vector(namely, comprising scFv (CLD18A2)-CAR).

The invention also provides a genetically modified T lymphocyte, whichis transducted by the present nucleic acid, the present recombinantplasmid comprising the above mentioned nucleic acid, or the viruscomprising the said plasmid. Conventional nucleic acid transductionmethods in the present field, including non-viral and viral transductionmethods, can be used in the present invention. The non-virus-basedtransduction method comprises electroporation method and transpositionmethod. Recently, Nucleofector nuclear transfection apparatus developedby Amaxa can directly introduce foreign gene into the nucleus to realizehigh-efficiency transduction of target gene. In addition, thetransduction efficiency of the transposon systems based on SleepingBeauty system or PiggyBac transposon is greatly improved compared withthat of common electroporation method, and it has already been reportedabout the combinative application of Nucleofector transfection apparatusand the Sleeping Beauty system (Davies J K., et al. Combining CD19redirection and alloanergization to generate tumor-specific human Tcells for allogeneic cell therapy of B-cell malignancies. Cancer Res,2010, 70(10): OF1-10.) Such method not only has relatively hightransduction efficiency but also can achieve site-directed integrationof target genes. In one embodiment of the invention, the T lymphocytetransduction method for realizing the modification of the chimericantigen receptor is based on virus such as retrovirus or lentivirus. Thesaid method has advantages such as high transduction efficiency, stableexpression of foreign genes, and can shorten the time for in-vitroculture of T lymphocytes to reach clinical scale. The transductednucleic acid is expressed on the surface of the transgenetic Tlymphocyte through transcription and translation. In vitro cytotoxityassay on various differently cultured tumor cells prove that the Tlymphocyte modified by the present anti-CLD18A2 chimeric antigenreceptor gene has highly specific tumor cell killing effect (also knownas cytotoxicity). Therefore, the present nucleic acid encoding thechimeric antigen receptor protein, the plasmid comprising the saidnucleic acid, the virus comprising the said plasmid and the transgenic Tlymphocytes transducted by the above nucleic acids, plasmids or viruscan be effectively used for tumor immunotherapy.

In one embodiment, the genetically modified T lymphocyte of theinvention expresses a chimeric antigen receptor on the surface thereof,wherein the chimeric antigen receptor is coded and expressed by anucleic acid of one of SEQ ID NOs: 15-18. In another embodiment, thetransgenic T lymphocyte surface of the present invention expresses achimeric antigen receptor, whose amino acid sequence is selected fromone of SEQ ID NOs: 19-22.

Since currently there is no report of the CAR T targeting CLD18A2, theinventors for the first time successfully discovered an immune effectorcell (eg, T lymphocyte) suitable for CAR modification from numeroustumor related genes, and successfully prepared CAR-modified immuneeffector cells. Thus, a brand new treatment means is provided for tumorssuch as pancreatic cancer and stomach cancer.

The embodiments of the present invention are further described belowwith reference to specific examples. It should be understood that theseembodiments are for illustrative purposes only and are not intended tolimit the scope of the invention. The experimental methods not speciallynoted of particular conditions are usually according to conventionalconditions, such as those described in J. Sambrook et. al., Eds,Molecular Cloning: A laboratory Manual, 3rd edition, Science PublishingHouse (2002), or the conditions recommended by the manufacturer.

Example 1. The Expression of Single-Chain Antibody Against CLD18A2

By repeatedly researching and analyzing, the inventors identifiedseveral scFv antibodies recognizing CLD18A2, for short, referred as 125,163, and 175.

125 (SEQ ID NO: 1 (nucleotide), 2 (amino acid)), 163 (SEQ ID NO: 3(nucleotide), 4 (amino acid)), 175 (SEQ ID NO: 5 (nucleotide), and 6(amino acid)) single-chain antibody sequences were synthesized bygenetic synthesis based on bridging PCR. The synthesized products weredigested by Nhe1/BamH1 (purchased from NEB), ligated in plasmid vectorpCMV-V5-Fc (the said plasmid fuses and expresses human antibody Fc atthe downstream of the multi-cloning site, hereinafter referred to asV5-Fc for short, purchased from Shanghai raygene biotechnology Co., LTD)digested by the same Nhe1/BamH1 via T4 DNA and was transformed into thehost bacterium TOP10. The clones were picked and positive clones wereidentified by PCR, and confirmed by sequencing. V5-scFv-125-Fc,V5-scFv-163-Fc, and V5-scFv-175-Fc eukaryotic expression plasmid wereobtained.

The above expression plasmids were used to transfect well grownHEK-293F, cultured at 37° C., 5% CO₂, 125 rpm on a shaking bed for 7days. They were centrifuged for 10 min at 4000 rpm, and precipitate wasremoved. The supernatant were collected and filtered with a 0.45 μmfilter film. The processed sample was purified by protein A affinitycolumn (purchased from GE), and eventually the purified single-chainantibody-Fc fusion protein scFv-125-fc (for short scFv-125), scFv-163-fc(scFv-163 for short), scFv-175-fc (scFv-175 for short) were obtained.The identification result is shown in FIG. 3.

Example 2. Construction of the Stable Expression Cell Line of CLD18A1 orCLD18A2

1. Construction of Expression Vectors of CLD18A1 and CLD18A2 andPreparation of Lentivirus

The complete coding sequences of CLD18A1 (GenBank: NM_016369) andCLD18A1 complete coding sequence (Genbank: nm_001002026) weresynthesized by genetic synthesis technology based on bridging PCR. Aflag tag(DYKDDDK) was inserted into the c-terminal, andMluI/SalI(purchased from NEB) were added at both ends of the synthesizedgene segments. The segments were double-digested by MluI/SalI, ligatedin plasmid vector pWPT (purchased from addgene) double-digested by thesame MluI/SalI via T4 DNA, and were transformed into the host bacteriumTOP10. The clones were picked and identified by PCR, and confirmed bysequencing. That correct lentivirus vector plasmid PWPT-CLD18A1,PWPT-CLD18A2 were obtained. The above plasmids and packing accessoryplasmids (pGag-pol, pREV, pVsv-g (all purchased from addgene)) wereco-transfected at a certain ratio to 293T cell. After 48 h and 72 h oftransfection, CLD18A1 and CLd18A2 virus solutions were collected,sub-packed, and stored at −80° C.

2. Establishment of Stable Exogenous Expression Lineage for CLD18A1 andCLD18A2 and Western Blot Assay

The above collected CLD18A1 or CLD18A2 virus solutions were added into293T cells in 6 cm dish respectively. After 72 hours, cells werecollected and were lysed by cell lysis solution. On the other hand,human stomach cancer BGC-823 (purchased from Shanghai cell library ofInstitution of Science of China, TCHu11) and NCI-N87 (purchased fromATCC, CRL-5822) were infected by CLD18A2 virus, respectively. After thecells grew to full, they were lysed by cell lysis solution. 40 μgprotein from collected lysed cells were subjected to SDS-PAGE gelelectrophoresis, and the gel was assayed by immunoblotting, stained withmouse anti-FLAG antibody (purchased from sigma Aldrich). After PBS wash,incubated together with goat anti-mouse antibodies labeled byhorseradish peroxidase (purchased from santa cluz), and colored usingECL reagent, and finally, developed.

The Western blot results showed strips with molecule weight of about 28kD in 293T cells transfected with CLD18A1 or CLD18A2 (i.e. 293T-CLD18A1,293T-CLD18A2) and BGC-823 and NCI-N87 cells transfected by CLD18A2 (i.e.BGC-823-CLD18A2, NCI-N87-CLD18A2), but no strips in the untransfectedempty cells (FIG. 4), indicating the successful construction of celllines exogenously expressing CLD18A1 and CLD18A2.

3. Experiment Steps of Flow Cytometry Analysis of the Binding Profile ofEach Cell Line with Anti-CLD18A2 Antibody

Using a fluorescence activated cell sorter (FACS) (BD company,FACSCalibur), the respective binding ability of single-chain antibodiesscFv-125, scFv-163 and scFv-175 with each of the following cell lineswere tested.

The specific method is as follows:

1) 293T, 293T-CLD18A1, 293T-CLD18A1, 293T-CLD18A2, BGC-823,BGC-823-CLD18A2, NCI-N87, NCI-N87-CLD18A2 tumor cells at exponentialgrowth phase were inoculated into 6 cm flat dish with a inoculation celldensity about 90%, and incubated overnight at 37° C. in incubator.

2) The cells were digested by 10 mM EDTA, and collected bycentrifugation at 200 g×5 min. The cells were resuspended in 1%phosphate buffer solution containing calf serum (NBS PBS) at aconcentration of 1*10⁶-1*10⁷/ml), and added into cytometric pipe at 100ul/pipe.

3) Centrifuged at 200 g for 5 min, and the supernatant were discarded.

4) The antibodies to be tested, scFv-125, scFv163 and scFv-175 wereadded, and simultaneously using unrelated antibodies as the negativecontrol with final concentration of antibody of 20 μg/ml, and 100 ul ofantibody in each pipe. Then they were left on Ice bath for 45 minutes.

5) Each pipe was added 2 ml 1% NBS PBS, centrifuged at 200 g for 5 mintwice.

6) Supernatant was discarded. 1:50 diluted FITC-labeled goat anti-humanantibody (from shanghai KangChen Bio-tech Inc), 100 ul per tube wasadded, and then put on ice bath for 45 minutes.

7) Each pipe was added 2 ml 1% NBS PBS, centrifuged at 200 g for 5 mintwice.

8) The supernatant was discarded, and the pellet was resuspended in 300ul 1% NBS PBS, detected by flow cytometry.

9) Flow Cytometer data analysis software WinMDI 2.9 was used to analyzethe data.

The flow cytometry results showed that the single-chain antibodyscFv-125 can not only bind to CLD18A1 stably expressed 293T cells butalso CLD18A2 stably expressed 293T cells (FIG. 5), indicating that thissingle-chain antibody lacks binding specificity for CLD18A2. Luckily,single-chain antibody scFv-163 and scFv-175 can specifically recognizethe 293T stably expressing CLD18A2, not bind to 293T cells stablyexpressing CLD18A1, which indicates that these two single-chainantibodies can specifically recognize CLD18A2. Furthermore, these twosingle-chain antibodies can also specifically recognize BGC-823 orNCI-N87 cell lines stably transfected with CLD18A2, but do not bind tothe BGC-823 or NCI-n87 cells not transfected with CLD18A2.

Example 3. Construction of Lentiviral Plasmids Expressing ChimericAntigen Receptor Proteins Encoded by the Nucleic Acids of the PresentInvention, and Virus Packaging

Table 1 explains the connection sequence of the exemplary chimericantigen receptors of the present invention, the connection may also beseen in FIG. 2.

TABLE 1 Chimeric Extracellular binding region-transmembrane antigenregion-intracellular signal region receptor 1-intracellular signalregion 2, etc CLD18A2-δZ scFv(CLD18A2)-CD8-CD3δzeta (negative control)CLD18A2-163-Z scFv(CLD18A2-163)-CD8-CD3 zeta CLD18A2-175-ZscFv(CLD18A2-175)-CD8-CD3 zeta CLD18A2-163-28BBZscFv(CLD18A2-163)-CD28a- CD28b-CD137(i.e. 4-1BB)-CD3 zetaCLD18A2-175-28BBZ scFv(CLD18A2-163)- CD28a-CD28b-CD137-CD3 zeta

1. Amplification of Nucleic Acid Fragments

(1) Amplification of scFv (CLD18A2-163, CLD18A2-175) Sequences

Using v5-scFv-163-fc plasmid as template, in the primer pair, theforward primer (SEQ ID NO: 7) comprises part of 2A sequence and thereverse primer (SEQ ID NO: 8) comprises part of CD8 hinge sequence. ScFv(CLD18A2-163) was obtained by PCR amplification. In the same way, usingthe v5-scFv-175-Fc plasmid as template, scFv (CLD18A2-175) was obtainedby PCR amplification, using primer pair wherein the forward primercomprises part of 2A sequence (SEQ ID NO: 9) and the reverse primer (SEQID NO: 10) comprises part of the CD8 hinge sequence.

(2) The Nucleic Acid Sequence of Other Parts of the Chimeric AntigenReceptor

Other part of anti-CLD18A2 chimeric antigen receptor protein exceptscFv(CLD18A2-163, CLD18A2-175) were obtained by PCR using the sequencesSEQ ID NOs: 18 and 21 disclosed in CN 201310164725.X.

Wherein the eGFP-F2A sequence was obtained by PCR amplification usingplasmid of SEQ ID NO:18 described in the patent application number201310164725.X as template and SEQ ID NOs: 11 and 12 as primer pair.

Obtaining CD8-CD3ζ (Z) and CD28a-CD28b-CD137-CD3ζ (28BBZ): CD8-CD3ζ(Z)and CD28a-CD28b-CD137-CD3ζ(28BBZ) fragments were respectively obtainedby PCR amplification using scFv(GPC3)-CD8-CD3ζ (SEQ ID NO:18 in patentapplication 201310164725.X) and scFv(GPC3)-CD28a-CD28b-CD137-CD3ζ (SEQID NO:21 in patent application 201310164725.X) as templates and SEQ IDNOs: 13 and 14 as primer pair.

SEQ ID NO: 18 in 201310164725. X is corresponding to SEQ ID NO: 23 inthe present invention.

The SEQ ID NO: 21 in 201310164725. X is corresponding to SEQ ID NO: 24in the present invention.

2. Splicing of Nucleic Acid Fragments

The eGFP-F2A nucleic acid fragment obtained as mentioned previously,scFv (CLD18A2-163) or scFv (CLD18A2-175) nucleic acid fragments of equalmolar, and CD8-CD3ζ (Z) or CD28a-CD28b-CD137-CD3ζ (BBZ) nucleic acidfragments of equal molar were subjected to three-fragment splicing asshown in FIG. 2 and PCR. The splicing conditions were as follows:predenaturation at 94° C. for 4 min; denaturation at 94° C. for 40 s;annealing at 60° C. for 40 s; extending at 68° C. for 140 s, 5 cycles,and then overall extension at 68° C. for 10 min. After addition of DNApolymerase and forward primer (SEQ ID NO:11) and reverse primer (SEQ IDNO: 14), the PCR amplification were done for 30 cycles, and theamplification conditions were: pre-denaturation at 94° C. for 4 min;denaturation at 94° C. for 40 s; annealing at 60° C. for 40 s; extensionat 68° C. for 140 s, for 30 cycles; and then overall extension at 68° C.for 10 min. The fragments obtained after the amplification are asfollows (Table 2):

eGFP-scFv(CLD18A2)-163-Z (SEQ ID NO:15, 19),

eGFP-scFv(CLD18A2)-163-BBZ (SEQ ID NO: 16, 20),

eGFP-scFv(CLD18A2)-175-Z (SEQ ID NO: 17, 21),

eGFP-scFv(CLD18A2)-175-BBZ (SEQ ID NO: 18, 22).

The results identification where shown in FIG. 6.

TABLE 2 The sequences in the present invention Sequence Description SEQID NO: 1 Nucleic acid sequence encoding CLD18A2 single chain antibody125 SEQ ID NO: 2 Amino acid sequence of CLD18A2 single chain antibody125 SEQ ID NO: 3 Nucleic acid sequence encoding CLD18A2 single chainantibody 165 SEQ ID NO: 4 Amino acid sequence of CLD18A2 single chainantibody 163 SEQ ID NO: 5 Nucleic acid sequence encoding CLD18A2 singlechain antibody 175 SEQ ID NO: 6 Amino acid sequence of CLD18A2 singlechain antibody 175 SEQ ID NO: 7~14 Primer sequences SEQ ID NO: 15Nucleic acid sequence encoding chimeric antigen receptor proteinCLD18A2-163-Z SEQ ID NO: 16 Nucleic acid sequence encoding chimericantigen receptor protein CLD18A2-163-28BBZ SEQ ID NO: 17 Nucleic acidsequence encoding chimeric antigen receptor protein CLD18A2-175-Z SEQ IDNO: 18 Nucleic acid sequence encoding chimeric antigen receptor proteinCLD18A2-175-28BBZ SEQ ID NO: 19 Nucleic acid sequence encoding chimericantigen receptor protein CLD18A2-163-Z SEQ ID NO: 20 Amino acid sequenceof chimeric antigen receptor protein CLD18A2-163-28BBZ SEQ ID NO: 21Amino acid sequence of chimeric antigen receptor proteinCLD18A2-175-ZSEQ ID NO: 22 Amino acid sequence of chimeric antigen receptor proteinCLD18A2-175-28BBZ SEQ ID NO: 23 scFv(GPC3)-CD8-CD3ζ (SEQ ID NO: 18 in201310164725.X) SEQ ID NO: 24 scFv(GPC3)-CD28a-CD28b-CD137-CD3ζ (SEQ IDNO:21 in 201310164725.X)

3. Lentiviral Plasmid Vector Construction Method

As an example, the vector system used for constructing the lentiviralplasmid vector of the present invention belongs to a third generationself-inactivating lentivirus vector system, which comprises threeplasmids, namely, psPAX2 encoding Gag/Pol protein and Rev protein(purchased from addgene); envelope plasmid PMD2.G encoding VSV-G protein(purchased from addgene); and recombinant expression vector encodingtarget gene CAR which is based on empty vector PWPT-eGFP (purchased fromaddgene).

In the empty vector pWPT-eGFP, the elongation factor-1 alpha (EF-1α)promoter regulates the enhanced green fluorescent protein (eGFP), whilein the recombinant expression vector encoding target gene CAR, theco-expression of eGFP and CAR is realized via ribosome skipping sequence2A (shortened as “F2A”) from food-and-mouth disease virus (FMDV). F2A isa core sequence from FMDV 2A (or referred to as “self-splicingpolypeptide 2A”), which has the “self-splicing” function of 2A, and canrealize the co-expression of both upstream and downstream genes. Due tothe high splicing efficiency, the highly balanced expression of upstreamand downstream genes and the short sequence of itself, 2A provided aneffective and feasible strategy for constructing polycistronic vectorfor gene therapy. In particular, this sequence is often used in theimmune therapy based on chimeric antigen receptor gene modified Tlymphocyte, to realize the co-expression of the target gene and GFP oreGFP, therefore CAR expression can be indirectly detected by detectingGFP or eGFP.

According to the present example, a lentivirus expression vector whichco-expresses eGFP and a specific CAR linked by F2A was constructed,collectively, referred to as pWPT-eGFP-F2A-CAR (FIG. 1). The target geneeGFP-F2A-CAR obtained in step 2 (see 1(2) in Example 3, and the elementfollowing F2A is called as CAR for short) was digested by restrictionendonucleases MluI and SalI, linked into a pWPT vector digested in thesame way so as to construct a lentiviral vector expressing each chimericantigen receptor. After the successfully constructed vector wasconfirmed by MluI and SalI digestive identification and sequencing, itcan be subjected to lentivirus packaging. As previously mentioned, theeGFP-F2A-CAR was transcribed into a mRNA, but finally into two peptidechains as eGFP and anti-CLD18A2 chimeric antigen receptor. In theguidance of the CD8a signal peptide, the anti-CLD18A2 chimeric antigenreceptor would locate on cell membrane.

The obtained vectors comprising target CAR are as follows:

pWPT-eGFP-F2A-scFv(CLD18A2)-163-Z;

pWPT-eGFP-F2A-scFv(CLD18A2)-163-BBZ;

pWPT-eGFP-F2A-scFv(CLD18A2)-175-Z;

pWPT-eGFP-F2A-scFv(CLD18A2)-175-BBZ.

4. Packaging Lentivirus by Plasmid Transfection of 293T

HEK-239T cells (ATCC: CRL-11268) cultured to 6-10 generations wereinoculated at a density of 6×10⁶ in 10 cm petri dish, incubated at 37°C., under 5% CO₂ overnight, preparing for transfection. The culturemedium was DMEM (purchased from PAA company) with 10% fetal calf serum(purchased from PAA company). The next day, the culture medium wasreplaced with serum-free DMEM at about 2 hours before transfection.

The transfection steps were as follows:

4.1 20 g empty plasmids pWPT-eGFP (mock control) or 20 μg individualtarget gene plasmid pWPT-eGFP-F2A-CAR, together with 15 μg of packagingplasmid PAX2 and 6 μg envelope plasmid pMD2.G were dissolved into 5004MillQ water, and uniformly mixed.

4.2 62 μL of 2.5M CaCl₂ (purchased from Sigma company) was addeddropwise, and mixed homogenously at 1200 rpm/min vortex,

4.3 Finally, 500 μL of 2×HeBS (80 mM NaCl, 10 mM KCl, 1.5 mMNa₂HPO4.2H₂O, 12 mM glucose, 50 mM Hepes (purchased from Sigma), pH7.05,sterilization with a 0.22 um filter) was added dropwise, and mixedhomogenously at 1200 rpm/min for 10 s.

4.4 Immediately added into the petri dish dropwise, slightly shaken,incubated at 37° C. and 5% CO₂. Cultured for 4-6 h, the culture mediumwas replaced with DMEM containing 10% fetal calf serum.

Transfection efficiency (ie, the proportion of cells with greenfluorescence) was observed the next day of the transfection, and ˜80%positive transfection efficiency indicated successful transfectionexperiment. After 48 h or 72 h of transfection, a 0.45 μm filter(purchased from Millipore Company) was used to filter and collect thevirus, and centrifuged by Beckman Optima L-100 XP ultracentrifuge at28,000 rpm, 4° C. for 2 hours, and the centrifuged supernatant wasdiscarded. The precipitate obtained by centrifugation was resuspendedwith Quantum 007 culture solution (purchased from PAA Company) at 1/10-1/50 volume of the stock solution, packed at 100 μL/pipe and frozen at−80° C., waiting for virus titration or infecting T lymphocyte.

5. Determination of Titer of the Lentivirus Packed with Mock oreGFP-F2A-CAR

At Day 1, 293T cells were inoculated to a 96-well culture plate at1*10⁵/ml, with 1004/well, incubated at 37° C. and 5% CO₂. The culturemedium is DMEM medium containing 10% fetal calf serum. At day 2, 504/well culture supernatant was discarded, and 504/well fresh culturemedium was added, which contains polybrene at a final concentration of 6μg/ml, and incubated at 37° C., 5% CO₂ for 30 min. 10 4/well of virusstock solution or 14/well of concentrated virus solution was added,diluted for 5 times, with 4 gradients, in duplicate. Incubated at 37° C.5% CO₂ After 48 hours of infection, the flow cytometry was used fordetecting eGFP preferably with positive rate at 5-20% of the cellnumber, calculated as titer (U/ml)=positive efficiency×dilutiontimes×100×10⁴. The titers of the above mentioned viruses packaged by thecalcium phosphate transfection method, comprising MOCK (i.e. the emptyvector control) and each eGFP-F2A-CAR were all at about 0.5-2*10⁶ U/mL.Virus titer measured after concentration was about 2×10⁷ U/mL.

Example 4. Recombinant Lentivirus Infection of CTL Cells

Human peripheral blood mononuclear cells (provided by Shanghai BloodCenter) were obtained from healthy human peripheral blood throughdensity gradient centrifugation method. CTL cells were obtained fromperipheral blood mononuclear cells by negative sorting method with CTLcell magnetic beads (purchased from Stem Cell Technologies), and thesorted CTL cells were subjected to flow cytometry to determine thepurity of the CTL cells. CTL positive rate ≥95% was preferred for nextoperation. Quantum 007 lymphocyte culture medium (purchased from PAAcompany) was added at a density of about 1×10⁶/ml for culturing, andmagnetic beads (Invitrogen company) coated with both anti-CD3 and CD28antibodies, at 1:1 cell:magnetic bead ratio, and recombinant human IL-2with a final concentration of 100 U/ml (purchased from Shanghai HuaxinHigh Biotechnology Inc.) were added for stimulating and culturing for 24h. Then the above recombinant lentivirus were used to infect the CTLcells at MOI≈5. The infected cells were subjected to passage at adensity of 5*10⁵/ml every other day, while recombinant human IL-2 with afinal concentration of 100 U/ml was supplemented to the lymphocyteculture medium.

At the 8^(th) day of culture, each different chimeric antigen receptorexpression in infected CTL cells was tested by flow cytometry. BecauseeGFP and CAR were co-expressed, the cells detected to be eGFP positivewere the positive cells expressing chimeric antigen receptors.Non-infected T lymphocytes were taken as negative control, and thepositive rate of the virus expressing different chimeric antigenreceptors infecting CTL cells were shown in table 3. The positive rateresults showed that lentivirus infection method can obtain CAR⁺ CTLcells with certain positive rate.

TABLE 3 CTL cells transfected with eGFP possitive rate the following CARof the CTL cells Mock(empty vector control) 56% CLD18A2-Z, fused andexpressing 51% 163 single chain antibody CLD18A2-28BBZ, fused andexpressing 54% 163 single chain antibody CLD18A2-Z, fused and expressing52% 175 single chain antibody CLD18A2-28BBZ, fused and expressing 55%175 single chain antibody

After respectively infected and packaged with different chimeric antigenreceptors, the CTL cells were passaged at cell density of 5*10⁵/ml andcounted, and IL-2 (final concentration of 100 U/ml) was added to thepassage cell culture solution. At 11th day of culture, there isamplification of about 20-40 times, indicating that the CTL cellsexpressing different chimeric antigen receptors have the ability toamplify in vitro, thus ensuring subsequent in-vitro toxicity tests andin-vivo experiments.

Example 5. In Vitro Toxicity Effect Assay for the Cells ExpressingChimeric Antigen Receptor

The materials used in the in-vitro toxicity experiment are as follows:

The 293T and gastric cancer cell lines as shown in table 4 were used astarget cells. The effector cells were CTLs that were in vitro culturedfor 12 days as verified in example 4, and confirmed by FACS that theywere chimeric antigen receptor expression positive (noted as CAR⁺,chimeric antigen receptor positive). The effect:target ratios, upondifferent conditions were 3:1, 1:1, and 1:3, the number of the targetcells was 10000/well. According to different effect:target ratio, eachgroup set for five repeated wells, and the average value in 5 repeatedwells was taken into count. The detection time was the 18th hour.

Wherein each experiment group and each control group were as follows:

Each experiment group: Each target cell+CTL expressing differentchimeric antigen receptors,

Control group 1: target cell with maximum release of LDH

Control group 2: Target cell with spontaneous release of LDH

Control group 3: effector cells with spontaneous release of LDH.

Detection method: Carried out with CytoTox 96 non-radioactivecytotoxicity assay kit (Promega company). The method was a detectionmethod based on colorimetric method, and can replace ⁵¹Cr release assay.CytoTox 96® assay quantitatively determines lactate dehydrogenase (LDH).LDH is a stable cytoplasmic enzyme, is released during cell lysis, whoserelease profile is substantially the same as the release profile of ⁵¹Crin radioactivity analysis. The released LDH would be in the culturesupernatant and can be detected by a 30-minute coupled enzyme reaction.In enzyme reaction, LDH can transfer a tetrazole salt (INT) into redFormazan. The red product is directly proportional to the number oflyzed cells. Refer to the instruction of CytoTox 96 non-radioactivecytotoxicity assay kit for details.

The cytotoxicity calculation formula is as follows:

${{cytotoxicity}\mspace{20mu}\%} = {\frac{\begin{matrix}{{{Experimental}\mspace{14mu}{group}} - {Control}} \\{{{group}\mspace{14mu} 2} - {{Control}\mspace{14mu}{group}\mspace{14mu} 3}}\end{matrix}}{{{Control}\mspace{14mu}{group}\mspace{14mu} 1} - {{Control}\mspace{14mu}{group}\mspace{14mu} 2}} \times 100\%}$

As specifically shown in table 4 and table 5, the present CTLsexpressing chimeric antigen receptor (fusion expressing single chainantibody 163 or 175) CLD18A2-Z CAR⁺ and CLD18A2-28BBZ CAR⁺ havesignificant killing effect on 293T cells with high expression ofCLD18A2, but not on the 293T cells expressing CLD18A1, which indicatesthat they can selectively kill the cells with CLD18A2. Furthermore, thepresent CTLs expressing chimeric antigen receptor CLD18A2-Z CAR⁺ andCLD18A2-28BBZ CAR⁺ have also significant killing effect on two gastriccancer cell lines BGC-823 and NCI-N87 with high expression of CLD18A2(see table 4 and table 5), and it showed effect:target ratio dependency,that is, the higher the effect:target ratio, the stronger thecytotoxicity. However, there was no cytotoxicity to BGC-823 and NCI-N87that do not express CLD18A2.

The data of the effect-target dependency further indicated that thepresent CTL of anti-CLD18A2 chimeric antigen receptor showed specificcytotoxicity to gastric cancer cells with high CLD18A2 expression.

Comparatively, the CTL transfected by MOCK plasmid (empty plasmid vectorpWPT-eGFP not carrying CLD18A2-CAR) showed quite low cytotoxicity toabove 3 cell lines with high CLD18A2 expression. The data ofcytotoxicity to cell lines with high CLD18A2 expression exhibitssignificant difference between the CTL transfected by MOCK plasmid andthe CTL expressing the present anti-CLD18A2 chimeric antigen receptor.

The above results showed that the chimeric antigen receptor constructedby choosing the single-chain antibody against CLD18A2 can selectivelykill target cells with high CLD18A2 expression. In addition, from thecytotoxicity data, CAR T of CLD18A2-28BBZ has stronger cytotoxicity tocells expressing CLD18A2 than CART of CLD18A2-Z.

TABLE 4 Cytotoxicity of CAR T cells expressing single-chain antibody 163CLD18A2-28BBZ CLD18A2-Z different different MOCK different effect:targetratio effect:target ratio effect:target ratio CYTOTOXICITY (%) 3:1 1:11:3 3:1 1:1 1:3 3:1 1:1 1:3 293T-CLD18A1 8.9 7.3 6.2 5.2 4.8 4.3 3.1 2.32.6 293T-CLD18A2 50 38.9 16.7 30.4 20.8 13.6 5.4 4.4 4.5 BGC-823-CLD18A262.7 44.7 16.1 38.9 25.8 10.5 5.7 4.8 4.3 BGC-823 5.8 5.5 4.8 5.2 4.65.5 2.8 3.5 3.8 NCI-N87-CLD18A2 61.3 52.5 13.6 42.8 26.3 7.7 6.1 5.2 4.7NCI-N87 4.6 5.1 5.9 3.6 3.5 4.5 3.9 4.2 3.1

TABLE 5 Cytotoxicity of CAR T cells expressing single-chain antibody 175CLD18A2-28BBZ different CLD18A2-Z different mock different effect:targetratio effect:target ratio effect:target ratio cytotoxicity (%) 3:1 1:11:3 3:1 1:1 1:3 3:1 1:1 1:3 293T-CLD18A1 8.6 7.3 6 5.4 4.8 2.7 2.4 1.51.4 293T-CLD18A2 57.5 41 15 35.2 25.7 13.2 5.6 2.3 1.6 BGC-823-CLD18A269.4 45.4 17.2 43.5 24.6 8.7 9.2 6.9 3.4 BGC-823 4.5 4.8 5.2 3.8 3.9 4.52.5 3.5 2.8 NCI-N87-CLD18A2 68.2 44.2 16.3 41.5 28.2 10.2 10.1 8.2 3.3NCI-N87 5.2 2.9 3.9 4.2 3.3 4.5 2.5 3.2 4.3

Discussion

At present, CAR T cells have become a potential therapeutic means.However, many tumors, such as gastric cancer, do not have reports aboutCAR T cell therapy. There is research showing that CLD18A2 may be aspecific marker of stomach tissue, therefore, it can also be atherapeutic target for tumor such as stomach cancer. However, atpresent, only the monoclonal antibody has been considered as thecandidate drug with CLD18A2 as the therapeutic target, and whether itcan be successfully used for the corresponding tumor treatment or not isnot yet known. Therefore, it is necessary to find new treatment means.Considering the tissue specificity of CLD18A2, the present inventioncontemplates that if targeted therapy can be performed with CAR T cells,a novel antitumor formulation can be expected. However, it is known thatthe CLD18A2 antigen is a tightly-connected protein, whether it cancontact with CAR T cells and induce the killing of the correspondingtarget cells is not known. In addition, as the protein spatialconformation is very crucial to the whole protein, many monoclonalantibody lose their antigen binding activity or specificity whenevolving into single-chain antibodies. Fortunately, the inventors foundthat two single-chain antibodies (163 and 175) retained the antigenbinding specificity of the monoclonal antibody. Further research showsthat the CAR T cells composed of these two single-chain antibodiesretained the selective killing effect on CLD18A2 positive cells. Theresults of the invention showed that CLD18A2 can truly be a CAR T celltherapy target; the CAR T cell against CLD18A2 is a novel candidatetumor treatment candidate means.

All documents mentioned in this disclosure are all incorporated hereinby reference, as if each document is individually referred to as areference. Furthermore, it should be understood that after reading theabove teachings of the invention, those skilled in the art can makevarious variations or modifications to the present invention, theseequivalent forms are also within the scope defined by the appendedclaims.

The invention claimed is:
 1. A method for inducing cytotoxic effectagainst CLD18A2 positive cells, comprising contacting said CLD18A2positive cells with immune effector cells having a chimeric antigenreceptor (CAR) expressed, said CAR comprising an extracellular bindingregion, a transmembrane region and an intracellular signal region,wherein said extracellular binding region specifically recognizesCLD18A2.
 2. The method of claim 1, wherein said extracellular bindingregion comprises an antibody which specifically recognizes CLD18A2. 3.The method of claim 2, wherein said antibody is a single-chain antibodyor a single-domain antibody.
 4. The method of claim 1, wherein saidextracellular binding region comprises an amino acid sequence having SEQID NO: 4 or SEQ ID NO:
 6. 5. The method of claim 1, wherein saidtransmembrane region has a sequence comprising the sequence oftransmembrane region and hinge region of CD8 or CD28.
 6. The method ofclaim 1, wherein said intracellular signal region sequence is selectedfrom the following: the intracellular signal region sequence of CD3ζ,FcεRIγ, CD27, CD28, CD137, CD134, or a combination thereof.
 7. Themethod of claim 1, wherein said immune effector cells comprise Tlymphocytes, NK cells or NKT cells.
 8. The method of claim 1, whereinsaid CLD18A2 positive cells are tumor cells.
 9. The method of claim 1,wherein said immune effector cells are transduced by a nucleic acidhaving a sequence of SEQ ID NO: 3 or SEQ ID NO:
 5. 10. The method ofclaim 9, wherein said immune effector cells are transduced by anexpression vector comprising said nucleic acid.
 11. The method of claim10, wherein said expression vector is derived from lentivirus plasmid.